The nuclear transcription factor NF-κB is a key mediator of the inflammatory response to cytokines such as TNFα and IL-1 by acting as a transcriptional activator of downstream proinflammatory genes. The N-terminal region of the NF-κB protein has a Rel-homology domain (RHD) that allows the protein to bind DNA, and also contains a nuclear localization sequence. In unstimulated cells, the NF-κB heterodimer is normally not found in the nucleus, it is sequestered in the cytosol by IκB, an inhibitor of κB that blocks the nuclear localization signal of NF-κB.
The dormant form of NF-κB can be quickly activated by a large number of extracellular signals, including mitogens, cytokines (such as TNFα and IL-1), viral proteins, antigens, phosphatase inhibitors and ultraviolet light. Binding of proinflammatory cytokines, for example, to their receptors results in the activation of IκB kinase (IKK) which then phosphorylates IκB on specific serine residues. This phosphorylation of IκB results in the rapid degradation of IκB by the ubiquitin-proteasome pathway. Once IκB is removed, NF-κB is free to translocated into the nucleus of the cell and transcriptionally activate its targeted genes.
The ubiquitin-proteasome pathway involves the covalent conjugation of cellular proteins with the small polypeptide ubiquitin. Thereafter, the conjugated proteins are hydrolyzed by a 26S proteolytic complex containing a 20S degradative particle called the proteasome. This multicomponent system is known to catalyze the selective degradation of highly abnormal proteins and short-lived regulatory proteins, such as IκB.
Therefore, IKK, the proteasome and ubiquitin represent important regulatory steps in the inflammatory response and are attractive targets for therapeutic intervention. Accordingly, there is a need to develop assays which permit the study of agents which may affect the activity of IKK, the proteasome and ubiquitin. Agents found to be useful for affecting the activity of IKK, the proteasome and ubiquitin are potentially valuable therapeutic agents for the treatment of NFκB-mediated diseases.